The long term goal of our work is to gain a structural and functional understanding of the mechanisms of gap junction regulation. Gap junctions, formed of proteins called connexins (Cxs), provide an intercellular pathway for the propagation of electrical/molecular signals, which are necessary for cellular differentiation, metabolic homeostasis, and in excitable tissue, electrical coupling. This type of communication permits individual cell events to synchronize into the functional response of an entire organ. Defects in human Cx genes that affect cell coupling are associated with a variety of inherited disorders (e.g. Charcot-Marie-Tooth disease and hereditary non-syndromic deafness). Genetic manipulations in mice have demonstrated the functional importance of Cxs in a variety of organs. Moreover, not only the presence but also the proper regulation of gap junctions is critical for homeostasis. For example, intracellular acidification leads to closure of gap junctions in all native tissues and exogenous expression systems tested. The study of pH-dependent regulation of gap junctions becomes even more relevant given that intracellular acidification is a major consequence of tissue ischemia. Acidification-induced uncoupling has an impact on the preservation of tissue surrounding the ischemic area. Therefore, we have chosen Cx43, the most widely expressed junction protein in the heart, brain, and other tissues, as our model system to study the structural regulation of Cxs. Our objective is to apply biophysical approaches to investigate intra-and intermolecular interactions that define the structural regulation of Cx43 during pH gating. We hypothesize that the Cx43 carboxyl terminal domain (CT) acts as a gating "particle" that, under the appropriate conditions (e.g. intracellular acidification or phosphorylation), binds to a "receptor" (i.e. Cx43 cytoplasmic loop;CL) affiliated with the pore and closes the channel. The following Specific Aims are proposed to investigate this concept: 1) To establish how the CT interacts with molecular partners that are involved in gap junction regulation;2) To assess the structural effect of pH on the CT and CL domains;3) To characterize cytoplasmic domain interactions between Cx isoforms. These Aims are designed to identify the functional consequences resulting from CT interactions with molecular partners and the CL in an effort to develop site-directed, specific modulators of gap junction communication with potential implications in therapeutic treatment of disease and ischemic injury.